Functional Analysis on a Naturally Occurring Variant of the Staphylococcus Aureus Uracil DNA Glycosylase Inhibitor

Papp-Kádár, Veronika; Balázs, Zoltán; Nagy, Gergely; Juhász, Tünde; Liliom, Károly; Vértessy, Beáta G.

doi:10.3311/ppch.10163

Cited by 2 publications

(4 citation statements)

References 16 publications

(29 reference statements)

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“…Previously the complex formation between wild-type SAUGI and the SAUGI E24H mutant has been partially investigated by microscale thermophoresis and isothermal titration microcalorimetry [19]. With regard to the dissociation constant determined for the SAUDG: SAUGI WT complex, it is of interest to note that our currently determined value obtained by mass spectrometry (14.4 AE 1.5 nM) compares more favorably to the dissociation constant of the same complex determined by surface plasmon resonance (1.2 nM) [17], as compared with the data obtained by isothermal titration microcalorimetry (131 AE 31 nM) [19]. It has been already observed that the lengthy microcalorimetry technique is not optimal for proteins that are sensitive to stirring, temperature and buffer/salt conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Protein expression and purification were performed as described previously . In brief, expression was performed in E. coli Rosetta BL21 (DE3) PlysS cells (Novagen, EMD Biosciences, Inc., Merck KGaA, Darmstadt, Germany) at 16 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Several inhibitory proteins can modulate catalytic activity of the UDG enzyme. At present, three different uracil‐DNA glycosylase inhibitor proteins have been described in the literature, namely Uracil‐DNA glycosylase inhibitor (UGI) , p56 and Staphylococcus aureus UGI (SAUGI) . The amino acid sequences of these inhibitory proteins are strikingly different; however, all of them present a protein surface mimicking the DNA negatively charged double helical structure .…”

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confidence: 99%

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Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

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The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA . The uracil base is among the most frequently occurring erroneous bases in DNA , and is cut out from the phosphodiester backbone via the catalytic action of uracil‐ DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil‐ DNA glycosylase. Interestingly, both uracil‐ DNA glycosylase ( Staphylococcus aureus uracil‐ DNA glycosylase; SAUDG ) and its inhibitor ( S. aureus uracil‐ DNA glycosylase inhibitor; SAUGI ) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil‐ DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI . Here, we investigated whether these mutations drastically perturb the interaction with SAUDG . To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil‐ DNA glycosylase inhibitor proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

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“…The rabbit skeletal myosin subfragment-S1 was a kind gift of Máté Gyimesi, Eötvös University, Budapest, Hungary. These proteins were expressed and purified as described previously [23,24,25]. The proteins were dialyzed against a buffer pH 7.5 comprising 20 mM HEPES, 100 mM NaCl and 1 mM TCEP.…”

Section: Methodsmentioning

confidence: 99%

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

et al. 2019

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EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.

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Functional Analysis on a Naturally Occurring Variant of the Staphylococcus Aureus Uracil DNA Glycosylase Inhibitor

Cited by 2 publications

References 16 publications

Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

Mass spectrometry‐based analysis of macromolecular complexes ofStaphylococcus aureusuracil‐DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

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